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Monday, August 29, 2011

Our immune system is capable of generating antibody against bacterial proteins located inside and outside of the bacterial cell. However only those antibodies targeting surface proteins (or other surface components) have the potential to eliminate the bacteria.

Unlike typical Gram negative bacteria, the external surface of the Treponema pallidum outer membrane is bare except for a tiny number of proteins protruding from the membrane. Most of the proteins targeted by the host antibody response are safely tucked away beneath the surface, inaccessible to the antibodies that recognize them. T. pallidum even lacks LPS, which is a major target found on typical Gram negative bacteria. The barren surface of T. pallidum is therefore one factor that may allow the "stealth pathogen" to persist in the host despite a strong antibody response.

None of the rare T. pallidum outer membrane proteins (Omps) have been identified with certainty, until now. Scientists at the University of Connecticut have finally confirmed that TP0326 is one of these rare Omps.1 Desrosiers and colleagues showed that the protein TP0326 was digested when proteinase K was added to live T. pallidum. Proteins known to be located in the periplasm were left untouched by the protease, indicating that the fragile outer membrane remained intact while the spirochetes were being harvested for the experiment. These results indicated that TP0326 was exposed on the surface of T. pallidum.

TP0326 (also called "Tp92") was first identified as a candidate Omp over a decade ago when rabbit antibodies against the protein were shown to promote opsonophagocytosis (engulfment) of T. pallidum.2 Antibodies must physically link bacteria to phagocytes for opsonophagocytosis to proceed. Opsonophagocytosis therefore occurs only when antibodies against surface-exposed proteins are present. The amino acid sequence of TP0326 also gave clues to its location. TP0326 was identified as a homolog of the Omp85 family,2 a set of proteins known to reside in the outer membrane of other Gram negative bacteria. Omp85 was later renamed BamA when it was shown to be the core component of the outer membrane protein complex "BAM" that inserts newly expressed Omps into the outer membrane.3

A number of computer programs predicted that TP0326 spanned the outer membrane as a β-barrel structure, which I described in an earlier post.4 The gallery of E. coli transmembrane Omps displayed below shows that the loops on one side of the barrel are exposed on the surface of the bacterium. The transmembrane portion of BamA is depicted as a box because its structure has yet to be determined experimentally, but it is also likely to have a β-barrel structure.

Figure 1 from Burgess 2008. The outer membrane is colored gray. The numbers indicate the number of β strands that cross the membrane. The periplasm is located below the outer membrane.

The amino terminus of BamA consists of five repeating structures called POTRA, which are thought to guide the β strands of new transmembrane Omps into the outer membrane.3 Note that the POTRA domains protrude into the periplasmic space. The amino terminus of TP0326 is thought to possess the POTRA domains as well.

Given the prediction that TP0326 structurally resembles BamA, it wasn't too surprising that TP0326 was exposed on the surface of T. pallidum. What was surprising was how TP0326 was targeted by the immune system in syphilis patients. Among the six patients examined by Desrosiers et al., three lacked any antibody whatsover against TP0326. Although the other three syphilis patients managed to generate antibody against TP0326, the antibodies reacted weakly (1 patient) or not at all (2 patients) with the β-barrel domain, which contained the surface-exposed loops. Instead, the antibodies targeting TP0326 reacted strongly with the subsurface POTRA domains in these three patients. Assuming that the results with these six patients can be extrapolated to other syphilis patients, humans infected with T. pallidum are incapable of generating a strong antibody response against the surface-exposed loops of TP0326.

Since the exposed regions of TP0326 appear to be an Achilles heel of T. pallidum, TP0326 may have evolved to avoid generation of antibodies targeting its vulnerable segments. Experimentally infected rabbits, which are not a natural host of T. pallidum, succeeded in generating antibody against the TP0326 β-barrel domain. Unfortunately the authors didn't present any data indicating whether the surface-exposed loops were recognized by the rabbit antibodies. However, as I mentioned above, earlier work showed that infected rabbits produce opsonic antibody against TP0326.2 The same paper showed that TP0326 was somewhat effective as a subunit vaccine in the rabbit model of syphilis.2 Both of these observations suggest that rabbits are able to produce antibody that reacts with the surface-exposed regions of TP0326. The rabbit model could be used to figure out conclusively whether the effectiveness of TP0326 as a vaccine relies upon generation of antibodies against the surface-exposed loops of the protein. If so, an approach for developing a syphilis vaccine would be to coax the human immune system into generating antibodies that target the surface-exposed loops of TP0326.

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Common Spirochete Diseases

Lyme disease is a tick-borne disease caused by several members of the Borrelia burgdorferi complex. B. burgdorferi, B. garinii, and B. afzelii account for most cases worldwide. A rash may appear at the site of the tick bite, and the patient may experience flu-like symptoms. Left untreated, the patient may suffer from neurologic, arthritic, and cardiac complications.

The syphilis agent Treponema pallidum is most commonly acquired by sexual contact. A skin lesion called a chancre appears at the site of initial contact with the spirochete. T. pallidum later spreads to other sites in the body to cause the flu-like symptoms and rash of secondary syphilis. Once secondary syphilis resolves, the spirochete may persist for years without causing problems. Later, tertiary syphilis can result in damage to vital tissues. Neurosyphilis and cardiovascular syphilis are two common forms of tertiary syphilis.

Leptospira lives in the kidneys of rodents and other reservoir hosts and is shed via urine into the environment. Humans acquire the spirochete by contact of abraded skin or mucous membranes with infectious urine or contaminated water or soil. Leptospirosis patients may initially experience flu-like symptoms. Jaundice and impaired kidney function occur in the potentially deadly form of leptospirosis called Weil's disease.